The overall goal of our studies is to better understand structure and function of HIV-1 integrase (IN) as a therapeutic target. A multimer of IN stably associates with two viral DNA ends in the context of the preintegration complex (PIC) and catalyzes concerted integration of the viral DNA ends into the host chromosome, a process which is essential for retroviral replication. Lens Epithelium Derived Growth Factor (LEDGF/p75) is a key cellular binding partner of HIV-1 IN and contributes to effective integration by tethering PICs to active genes. During the past funding cycle the efforts in the PI's group focused on examining molecular interactions of IN with viral DNA substrates, LEDGF/p75 and small molecule inhibitors. We have mapped HIV-1 IN interactions with viral DNA and identified novel protein contacts essential for the 3'-processing reactions. Furthermore, our group has uncovered new inter- and intra-protein-protein contacts for the full length IN-LEDGF/p75 complex and demonstrated that the cellular cofactor strongly modulates highly dynamic structure and function of the retroviral enzyme. Moreover, our group identified a small molecule inhibitor of HIV-1 IN that selectively binds to the IN dimer interface. Collectively, these findings have laid a strong foundation for extending our efforts further to address the following new and important questions. Aim 1 will identify functional contacts between IN, viral DNA and LEDGF/p75 in concerted integration intermediates using our MS-based footprinting approach. Aim 2 will examine modulation of IN structure and function by LEDGF/p75 using protein-protein FRET approach. Aim 3 will probe potential direct interactions between LEDGF PWWP and the modified histones using a histone peptide array. Aim 4 will further investigate the mechanism of action for the small molecule allosteric inhibitor of IN by analyzing various derivatives of the lead compound. Taken together, these new findings will enhance our understanding how LEDGF/p75 modulates IN interactions with viral DNA and navigates the IN-viral DNA complex to the active transcription units at the chromatin. Furthermore, our studies with the allosteric inhibitor will contribute to wider efforts in the field focused on discovery of new antiretroviral therapies. PUBLIC HEALTH RELEVANCE: HIV-1 integrase (IN) is commonly viewed as an important therapeutic target for the following reasons: its catalytic activities are required for the virus life cycle, there is no closely related human equivalent of IN, and specific IN inhibitors are likely to be effective against viral strains resistant to currently available therapies targeting HIV-1 reverse transcriptase, protease and fusion. The proposed studies will investigate molecular interactions of IN with viral DNA substrates, the key cellular cofactor LEDGF/p75 and small molecule inhibitors. The obtained results will improve our understanding of how this key retroviral enzyme functions and contribute to wider efforts in the field focused on discovery of new antiretroviral therapies. 1